The initiation of blood clotting is by two different, yet similar, molecular mechanisms called the intrinsic and extrinsic coagulation pathways, or cascades. The intrinsic pathway involves factors that are normally found in the blood. The extrinsic pathway involves tissue factors in addition to blood components. In each of the reaction steps of the two cascades, a proteinase converts an inactive zymogen into its enzymically active form. In the last step of the cascade, which is the same in both the intrinsic and extrinsic pathways, inactive prothrombin is converted to thrombin, which in turn catalyzes the conversion of soluble fibrinogen into insoluble fibrin.
Factor IX participates in the cascade of events that lead to blood coagulation. Specifically, Factor IX, when activated by the action of Factors XI.sub.a or VII.sub.1, activates Factor X to X.sub.a. Factor X.sub.a in turn activates Factor II (prothrombin) to Factor II.sub.a (thrombin). The activated Factor II then activates fibrinogen to form the fibrin polymers of the blood clot. A deficiency in the activity of any of the factors involved in blood clotting leads to an inability of the blood to clot properly or to longer-than-normal clotting times. For example, Factor IX is absent or deficient in patients who have a condition identified as "Hemophilia B." Thus, the blood of Hemophilia B patients does not clot properly. Factor IX is administered to Hemophilia B patients to provide sufficient Factor IX, to return the clotting ability of their blood to as close to normal as possible.
Commercially available Factor IX concentrates frequently include other blood factors in addition to Factor IX. For example, some such preparations comprise the prothrombin complex which includes Factors II, V, and X in addition to Factor IX.
The occurrence of thrombotic complications, such as deep vein thrombosis, disseminated intravascular coagulation, and pulmonary embolism have been reported in patients treated with prothrombin complex concentrates or in Factor IX preparations that are contaminated with Factor II and/or Factor X. These complications are frequently seen in premature infants, in patients with poor liver function, and in surgery patients. Such complications have also been observed in Hemophilia A patients receiving prothrombin complex concentrate as a Factor VIII inhibitor bypassing agent.
The thrombogenic component of prothrombin complex concentrates has been attributed most often to either activated factors, coagulant active phospholipid, or zymogen overload. Zymogen overload may be the basis of disseminated intravascular coagulation in surgical situations where patients receive large and repetitive doses of prothrombin complex concentrates. In such cases, a buildup of zymogens in the circulation, particularly of Factors II and X, is likely to occur due to their relatively long half-life in relation to Factor IX.
The thrombotic complications associated with the use of prothrombin complex make it desirable to provide a Factor IX concentrate, essentially free of other proteins, for use in treating Hemophilia B patients
Various methods for enhancing the purity of Factor IX concentrates have been reported. For example, processes for producing concentrates of Factor IX, essentially free of prothrombin, and of Factor X by use of affinity chromatography on a sulfated dextran resin have been disclosed (D. Menache et al., "Coagulation Factor IX Concentrate: Method of Preparation and Assessment of Potential In Vivo Thrombogenicity in Animal Models", Blood, 64. 1220-1227 [1984]). Factor IX has been purified by affinity chromatography on a heparin-sepharose resin (L-O. Andersson et al., "Purification and Characterization of Human Factor IX", Thrombosis Research, 7, 451-459 [1975]). Factors IX and X have been separated by using a process which includes heparin-agarose chromatographic techniques (S. P. Bajaj et al., "A Simplified Procedure For Purification of Human Prothrombin Factor IX and Factor X", Preoarative Biochemistry, 11, 397-412 [1981]). Procedures are a)so known in the art for separating Factor IX by affinity chromatography on a dextran sulfate-sepharose gel.
While Factor IX can be separated on sepharose (agarose gels) in the laboratory, the use of agarose gels for large-scale separations has been found to be unsatisfactory. When the agarose gels are packed into commercial-size columns, they compress to an undesirable extent and thereby inhibit flow of liquids through the column. This problem has been overcome by the use of dextran sulfate silica resin, as described in U.S. Pat. No. 4,725,673 to Herring, incorporated herein by this reference. While this purification method is desirable in that the silica gel results in higher flow rates and, therefore, faster purification procedures, it uses a heat treatment to inactivate any viral contaminants that may be present in the human blood-derived protein preparations The heat treatment results in denaturation of a portion of the Factor IX which can lead to low specific-activity Factor IX preparations.
In addition to the above methods, purification of Factor IX has been performed using immunoaffinity and ion-exchange chromatography (S.S. Ahmad et al., "Rapid Purification of Factor IX, Factor X and Prothrombin by Immunoaffinity and Ion Exchange Chromatography", Thromb. Res., 55, 121-133 [1989]), which has resulted in specific-activities as high as 269 units/mg, for Factor IX. While immunoaffinity methods lead to high specific-activity preparations, the necessity to prepare monoclonal antibodies against the proteins to be purified adds a significant cost to the purification procedure.
It is therefore desirable to provide, at a relatively low cost, a process for the purification of Factor IX which yields a high specific-activity Factor IX preparation that is safe for use in humans.